Properties of the tobacco mosaic virus intermediate length RNA-2 and its translation

The existence of subgenomic RNAs is well established in the case of plant viruses such as tobacco mosaic virus (TMV). However, except for the subgenomic coat protein mRNA, it is not known whether the other subgenomic RNAs have a function in the life cycle of the virus. In search of more information about one of the major subgenomic RNAs-intermediate length RNA-2 or I2 RNA-of TMV, in vitro and in vivo translational studies were performed. The I2 RNA, which codes in vitro for the synthesis of a 30K (K = kilodalton) protein, appears to be uncapped as judged by the need of different in vitro translation conditions for the synthesis of this protein, compared to the conditions required for the synthesis of the 126K and 183K proteins coded by the capped genomic RNA. In vivo a protein migrating in the same position as the 30K protein synthesized in vitro can be detected in infected tobacco leaves. Since this protein occurs transiently early upon infection, whether it is virus-coded or virus-induced, it could have an early function during infection.     

Recombination in adenovirus: DNA sequence analysis of crossover sites in intertypic recombinants

The nucleotide sequence of the adenovirus type 5 genome has been determined for a 620-bp region that spans the C terminus of the pVI gene and the N terminus of the hexon gene, and compared to the adenovirus type 2 DNA sequence: 25 base changes have been identified, most of which do not lead to alterations in the amino acid sequence and regulatory signals in the region. Crossover sites in three intertypic recombinants have been previously located in this region of the genome by fine restriction mapping. A sequence determination for the three recombinants, and the four ts mutants used in generating the ts+ recombinants, was carried out. The crossovers were in each case located in a small region of complete sequence homology (from 45 to 156 nucleotides long) flanked on either side by sequences derived from each parent. These structures are compatible with a reciprocal crossing over model of generalised recombination, where a recombinant joint has resolved in a region of high DNA homology. For the recombinants considered here, this region abutts onto a neighbouring region of much lower sequence homology, and it is possible that the position of the crossover is determined at least in part by the termination of branch migration at a heterologous boundary.     

Role of lipid peroxidation in tissue injury after hepatic ischemia

Ischemia and anoxia are associated with decreased concentrations of cellular antioxidants. The hypothesis that recirculation of oxygenated blood to previously ischemic tissue may result in enhanced free-radical reactions leading to lipid peroxidation and tissue damage was investigated. Elevated hepatic conjugated diene concentrations were detected 60 min after treatment of rats with carbon tetrachloride, a positive control, but were not found after 90 min ischemia or at 5 or 60 min after reperfusion of ischemic tissue. These findings suggest that lipid peroxidation may not be an early event in ischemia-induced necrosis but do not rule out a role of other free-radical reactions in the pathogenesis of ischemic necrosis.     

Protease of adenovirus type 2: partial characterization.

An adenovirus-associated protease activity specific for the cleavage of core polypeptide PVII to polypeptide VII was identified and its properties were studied using an in vitro assay system. All temperature-sensitive (ts) mutants examined failed to induce protease activity at 39°. Activity was restored in revertants. The protease activity was completely inhibited by 1 mM tosylamide phenylethylchloromethyl ketone while phenylmethylsulfonylfluoride and tosyl-lysinechloromethyl ketone at 1 mM reduced enzyme activity to 36 and 10% of control, respectively. By contrast ethylenediamine tetraacetic acid had no effect. Optimum enzyme activity was observed at neutral pH. Enzyme activity was stable up to, but not beyond, 45°. Polypeptide PVII was cleaved whether it was in the soluble form, bound form, heat-, or acid-precipitated form. Wild-type young virions contain endogenous protease activity while the virions produced at 39° by tsl-infected cells do not. The PVII contained in these tsl-39 virions, however, may be processed by exogenous WT enzyme after the particles have been frozen-thawed several times. These results suggest that the adenovirus-associated protease is a chymotrypsin-like, nonmetalo, neutral protease.     

Rous sarcoma virus p19 binds to specific double-stranded regions of viral RNA: effect of p19 on cleavage of viral RNA by RNase III.

The extent of binding of purified RSV(Pr-C) p19 and p12 to a variety of RNAs was measured using a sensitive nitrocellulose filter binding assay which is capable of detecting binding reactions with association constants as low as 3 × 10⁶ liters × mol^?1 (Hizi, A., Leis, J. P., and Joklik, W. K. 1977). RSV p19 bound 60 and 34 S RSV (Pr-C) RNA with association constants of 5.1 × 10¹¹ and 1.8 × 10¹⁰ liters × mol^?1. RSV p19 bound preferentially to specific double-stranded regions of the RNA since: (a) The association constant for Neurospora nuclease-digested 34 S RNA was the same as for untreated RNA; (b) the association constant for 34 S RNA partially digested with Escherichia coli RNase III (which is specific for double-stranded RNA regions) was 30-fold lower than for untreated RNA; (c) p19 prevented cleavage of 34 S RSV-RNA by E. coli RNase III; (d) p19 bound cell precursor RNAs containing RNase III-sensitive sites, but not mature RNAs lacking RNase III-sensitive sites. On the other hand, purified RSV p12 bound all RNAs tested with association constants roughly proportional to their molecular weights. A possible function for p19 in regulating the processing of viral RNA and its subsequent translation has been proposed.     

Murine sarcoma viruses: the helper-independence reported for a Moloney variant is unconfirmed; distinct strains differ in the size of their RNAs.

A variant of Moloney murine sarcoma virus (Mo-MSV) reported to behave like a nondefective sarcoma virus was subjected to biological and biochemical analyses to determine whether its alleged helper-independence could be confirmed. When plated at low multiplicity the virus was shown to readily generate (four out of six) transformed clones which failed to produce virus unless superinfected with helper leukemia virus. The RNA of the parental virus stock was compared electrophoretically to that from clones which produced virus after the initial infection (producer clones) or after superinfection with Mo-murine leukemia virus (MLV) (nonproducer clones). All clones contained a MSV-specific 30 S RNA species. In addition, virus from one producer clone also contained 38 S MLV RNA at a high relative concentration, indicating that the original Mo-MSV stock must have contained such an RNA species. However, the original virus stock as well as virus from another producer clone contained 38 S MLV RNA at a low, uncertain relative concentration. A hypothesis consistent with these and previous data suggests that the Mo-MSV variant investigated here is defective and contains helper leukemia virus at a low concentration. This explains (i) the ready generation of nonproducer clones by infection at low multiplicity, (ii) the difficulty in detecting helper leukemia virus 38 S RNA in the original virus stock, and (iii) the low complexity (approximately 1.9 × 10⁶ daltons) of the MSV-specific 30 S RNA. These results are compatible with the properties reported for a defective MSV genome, but incompatible with those of a nondefective MSV genome. The MSV-specific RNA components of different clonal isolates of Mo-MSV differed from each other in size, ranging between 2.1 and 1.6 × 10⁶. The Harvey sarcoma virus-specific RNA was 1.9 × 10⁶, that of Kirsten sarcoma virus was 2.5 × 10⁶, and the spleen focus forming component of Friend virus was 2.0 × 10⁶. The sarcoma- or transformation-specific RNA components of all transforming viruses tested here were smaller than the 38 S RNA of helper leukemia viruses of 3.1 × 10⁶.     

High incidence of horse serum protein allergy in various autoimmune disorders.

In 186 persons (68 patients suffering from different so-called autoimmune diseases, 30 kidney recipients, 38 control patients from a surgical ward, and 50 healthy volunteers) the immune response to horse IgG was examined. The lowest rate of sensitization was found in kidney transplant recipients (3%) and the highest in autoimmune patients (33%). After excluding 39 patient who had received horse serum treatment prior to the examination, it was found that without previous injection of horse serum, 27% of the patients with autoimmune disease were sensitized to horse IgG. Compared to the other groups (kidney transplant recipients, 4%: surgical controls, 0%; healthy volunteers, 3%), this difference was statistically significant (p is less than 0.01).     

Angiomyolipoma mimicking true lipoma of the liver: report of two cases

Hepatic angiomyolipoma (AML) is very rare and only about 80 cases have been reported. The tumor is fundamentally heterogeneously composed of the three tissue components of blood vessels, smooth muscle cells (SMC), and fat cells. Two cases of hepatic AML are reported here, both of which are histologically composed predominantly of a fat cell element and resembled true lipoma (lipomatous AML). However, careful examination of both tumors revealed the presence of a small amount of epithelioid SMC, especially around blood vessels. Immunohistochemical study using monoclonal antibody for melanoma (HMB-45) clearly revealed a small amount of HMB-45-positive SMC around the blood vessels and scattered in the diffuse fat cell growth in both tumors. Since no liver tissue components or primary liver tumors are reactive with HMB-45 except AML cells, the presence of HMB-45-positive cells within the tumor clearly established the diagnosis of hepatic AML. Any fatty tumor or focal fatty lesion of the liver that superficially resemble true lipomas should be tested for the presence of HMB-45-positive SMC in the tumor to differentiate it from AML.     

Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: Effect of FK506, an inhibitor of T cell activation

Objective and design: To characterize rat collagen-induced arthritis (CIA) on the basis of levels of inflammatory cytokines, tumor necrosis factor (TNF)-, interleukin (IL)-1 and IL-6 in paw tissues, and further investigate the effect of FK506 (tacrolimus), a potent inhibitor of T cell activation, on cytokine levels.Methods: CIA was induced in female Lewis rats. The volume of hindpaws was measured before and after collagen immunization. TNF-, IL-1 and IL-6 levels in paw tissue extracts were determined by ELISA. Proteoglycan contents of cartilage in femoral heads was measured as an indication of cartilage destruction. To assess the effect of FK506 on inflammatory cytokine levels, rats were orally treated with 5 mg/kg of FK506 from days 14–21.Results: TNF- a level in paw tissues did not significantly change compared to levels found before collagen immunization, throughout development of CIA. In contrast, IL-1 and IL-6 levels in paw tissues significantly increased between day 14 and day 28 after collagen imuninization, when the arthritis was at a developed stage. Therapeutic treatment with FK506 reduced the elevated level of IL-6, but not IL-1, in paw tissue. FK506 treatment was effective in suppressing paw swelling and also recovering the loss of proteoglycan contents in the cartilage.Conclusions: Levels of IL-1 and IL-6, but not TNF- , in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1 and IL-6, rather than TNF- , may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6.Received 4 March 2004; returned for revision 2 April 2004; accepted by M. J. Parnham 9 April 2004